The present invention relates to nucleic acid molecules and constructs containing these nucleic acid molecules and methods of using these constructs to express heterologous genes in hosts. In particular, the nucleic acid molecules of the present invention comprise a DNA sequence encoding at least a portion of the xcex1 subunit of the RNA polymerase (referred to herein as RNAP) obtained from the same genus source from which the heterologous genes were originally isolated. The nucleic acid constructs also can optionally comprise, if required for expression of the heterologous genes, at least one gene encoding a transcriptional regulator, also obtained from the same genus as the source of the heterologous genes.
Although many host cell systems are well characterized and utilized to express a number of heterologous genes, there are still many genes that cannot be expressed in such host cell systems, as for example, Escherichia coli (E. coli). It is believed that the lack of expression in these host cell systems is a result of the promoter sequences of the heterologous genes not being recognized by the host RNAP. The present invention has solved this problem by showing that this block in expression can be overcome by co-expression of at least a portion of the rpoA gene product, the gene that encodes the xcex1 subunit of the RNAP, obtained from the same genus as the source of the heterologous gene that is desired to be expressed in the new host cell. The co-expressed xcex1 subunit of the RNAP from the same genus as the source of the heterologous gene combines with the other subunits of the RNAP, xcex2, xcex2xe2x80x2, and "sgr", to form a functional RNAP. Additionally, if transciptional regulators, such as transcriptional activators or transcriptional repressors, or additional other subunits of the RNAP, such as xcex2, xcex2xe2x80x2 or "sgr", are required to obtain expression of the heterologous gene, then these additional components also are obtained from the same genus as the source of the heterologous gene. Since two different rpoA genes are present in a single host cell, there will be various combinations of RNAPs present in the host cell (1) RNAP containing two xcex1 subunits of the same genus as the host (2) RNAP containing two xcex1 subunits from the same genus source as the heterologous gene, and (3) RNAP containing one xcex1 subunit from the same genus as the host and one xcex1 subunit from the same genus source as the heterologous gene. A heterologous gene is intended to mean a gene that is not from the same source as the host cell.
Agrobacterium tumefaciens is a Gram-negative soil bacterium which is the causative agent of Crown Gall disease, affecting primarily dicotyledonous plant species (reviewed in 18, 62). The pathogen incites production of the characteristic tumor through the transfer of a piece of DNA (T-DNA) from the Ti (Tumor inducing) plasmid into susceptible plant cells, with subsequent integration into the host genome. The T-DNA contains genes that direct the biosynthesis of auxin and cytokinin in infected cells (1, 57), resulting in uncontrolled cell division leading to production of the characteristic tumor. The T-DNA also contains genes for the biosynthesis of unique compounds called opines which the bacterium can utilize as a carbon and nitrogen source (39).
Successful transfer of the T-DNA is dependent on the coordinated expression of virulence (vir) genes located on the Ti plasmid but separate from the T-DNA. Expression of vir genes occurs in response to certain phenolic compounds released from wounded plants (54). This expression is augmented by monosaccharides (5, 52), and an acidic pH (38) which are characteristics of plant wound sites. Expression of vir genes requires virA and virG, which are members of the family of two component regulatory systems (60). VirA is an inner membrane associated histidine protein kinase which autophosphorylates in response to the environmental signals (19, 28). The phosphate moiety is subsequently transferred to the aspartate residue of VirG, which in turn activates transcription from promoters containing a specific 12 base pair sequence called the vir box, present in the promoters of all vir genes (29, 44). In addition to virA and virG, other chromosomally encoded genes have been identified in A. tumefaciens that have been shown to modulate virulence gene expression either directly or indirectly (12, 15, 20, 61).
The use of E. coil as a heterologous host in which to study the regulation of A. tumefaciens virulence genes and the mechanism of T-DNA transfer constitutes an ideal model system given the degree of characterization at both the biochemical and genetic level. However, all previous attempts to reconstitute vir gene expression in E. coil have not been successful. Possible explanations for the lack of vir gene expression include the presence of unidentified regulatory genes in A. tumefaciens required for vir induction, and/or that E. coli may contain specific repressor(s) of vir gene induction.
A characteristic of vir gene promoters is the absence of a strong xe2x88x9235 sequence (10). Dnase I footprinting studies have shown that VirG protects a region extending into where the xe2x88x9235 consensus sequence should be (29, 44). It has been suggested that binding of VirG may functionally replace the xe2x88x9235 consensus sequence allowing transcription to occur. This situation is similar to Class II CAP-dependent promoters, in which the CAP binding site overlaps with the xe2x88x9235 sequence. Studies have demonstrated that transcription at Class II CAP-dependent promoters requires interaction between CAP and the xcex1 subunit (rpoA) of RNAP (42, 49, 64). Many transcriptional factors from E. coli are known to require interaction with the xcex1 subunit of RNAP, including FNR (59), GaiR (9), MarA (24), Mer R (7), MetR (23) OxyR (56), OmpR (21), Rob (26), SoxS (25), and TyrR (34). Analysis of the xcex1 subunit from E. coli indicates the presence of two independent domains, the N-terminal domain and the C-terminal domain (21, 27, 63). The N-terminal domain (NTD) is involved in the assembly of the core polymerase, while the C-terminal domain (CTD) is involved in interaction with certain transcriptional regulators (7, 9, 21, 23, 24, 25, 26, 32, 34, 47, 56, 59). Recently, interaction between the NTD and CAP at Class II CAP-dependent promoters has been demonstrated (42,49).
The present methods of expressing heterologous genes are particularly useful to express multiple genes or operons in a metabolic pathway by co-expressing the rpoA gene product obtained from the same natural hosts from which the genes or operons were originally isolated with the multiple genes. The present method provides an advantage over existing multiple gene expression systems by eliminating the need to separately isolate each gene in the metabolic pathyway and link each gene to a promoter that is functional in the host. Expression of heterologous genes for an entire metabolic pathway in hosts is particularly useful to produce particular metabolites, such as pharmaceuticals, food supplements, chemical products or fine chemical products.
The present invention further provides nucleic acid molecules and methods whereby heterologous genes encoding metabolites in an entire metabolic pathway can be introduced into hosts so that these hosts possess specific catabolic and/or metabolic characteristics that allow the hosts to grow or express gene products under specific environmental conditions. These hosts are useful in bioremediation, bioassays and biocontrol studies where manipulation of natural or symbiotic bacterial flora is desired, such as in the control of parasites or insects where bacterial symbiants are involved.
The present invention broadly relates to the expression of heterologous genes in a host using nucleic acid molecules comprising a gene encoding the complete xcex1 subunit of the RNAP obtained from the same genus as the source of the heterologous genes or at least a portion of the xcex1 subunit of the RNAP. The host and the heterologous genes to be expressed may be prokaryotic or eukaryotic in origin, however, prokaryotic hosts are preferred. The present invention also relates to a hybrid nucleic acid molecule encoding a hybrid xcex1 subunit of RNAP, where a first nucleic acid comprises at least a portion of the rpoA gene encoding the xcex1 subunit of an RNAP obtained from the same genus as the source of the host cell and a second nucleic acid comprises at least a portion of the xcex1 subunit of an RNAP obtained from the same genus as the heterologous gene.
The present invention specifically discloses the rpoA gene from Agrobacterium encoding the xcex1 subunit of the RNAP and the corresponding amino acid sequence. Portions of both the rpoA gene and the xcex1 subunit of RNAP of Agrobacterium also are encompassed by the present invention, particularly the sequences disclosed in FIGS. 2A (SEQ ID NO:1) and 2B (SEQ ID NO:2). The rpoA gene is useful in methods of expressing Agrobacterium genes in hosts, particularly prokaryotic host cells. Prior to the present invention, it was not possible to express Agrobacterium genes, particularly Agrobacterium virulence genes, in E. coli. Therefore, the present invention provides a method for studying Agrobacterium genes and their regulation.
The invention also relates to genes encoding hybrid rpoA genes that comprise at least a portion of an rpoA gene from the same genus as a heterologous gene which is expressed, such as the Agrobacterium rpoA gene, with the remainder of the rpoA gene obtained from the same genus as the host cell in which the heterologous gene is expressed.
The present invention is directed to an isolated nucleic acid molecule encoding the complete xcex1 subunit of an RNAP of Agrobacterium or at least a portion thereof. Specifically, the present invention is directed to an isolated nucleic acid molecule encoding the complete xcex1 subunit of an RNAP of Agrobacterium, in which the amino acid sequence of the xcex1 subunit is depicted in. FIG. 2A (SEQ ID NO:1) or at least a portion of this sequence. More specifically, the present invention is directed to an isolated nucleic acid molecule comprising the complete nucleic acid sequence as depicted in FIG. 2B (SEQ ID NO:2) or at least a portion thereof. These isolated nucleic acid molecules encoding at least a portion of the xcex1 subunit of an RNAP of Agrobacterium also are optionally linked to a promoter that is functional in the host in which these molecules are expressed. Further, the present invention is directed to vectors containing these nucleic acid sequences or molecules. The vectors are useful in transforming host cells for the expression of heterologous gene(s). The invention is also directed to host cells that are transformed with the nucleic acid molecules described herein. The nucleic acid molecules useful to transform the host cells are operably linked to a promoter and encode an xcex1 subunit of an RNAP or at least one heterologous protein.
The present invention is also directed to a hybrid nucleic acid molecule for the expression of at least one heterologous gene in a host cell comprising a first nucleic acid sequence encoding at least a portion of the xcex1 subunit of an RNAP obtained from the same genus as the host cell and a second nucleic acid sequence encoding at least a portion of the xcex1 subunit of an RNAP obtained from the same genus as the source of the heterologous gene; and vectors containing the hybrid nucleic acid molecule.
The present invention is further directed to a method of expressing at least one heterologous gene in a host cell comprising transforming a host cell with a vector comprising a nucleic acid molecule encoding a complete xcex1 subunit of RNAP from the same genus as the source of the heterologous gene or at least a portion thereof, and also transforming the host cell with a vector comprising at least one heterolgous gene. The nucleic acid molecule and the heterologous gene(s) also may be contained in one vector but preferably these sequences are contained within two or more vectors. In a further embodiment, the present invention is also directed to a method of expressing at least one heterologous gene in a host cell comprising transforming a host cell with a vector comprising a hybrid nucleic acid molecule comprising a first nucleic acid sequence encoding at least a portion of the xcex1 subunit of an RNAP obtained from the same genus as the host cell and a second nucleic acid sequence encoding at least a portion of the xcex1 subunit of an RNAP obtained from the same genus as the source of the heterologous gene, and transforming the host cell with a vector comprising at least one heterologous gene; and culturing the transformed host cell under conditions where the heterologous gene is expressed in the host cell. The host cell also may be transformed with a single vector containing the nucleic acid molecule encoding the complete xcex1 subunit of the RNAP, a portion thereof or a hybrid molecule comprising a portion of the xcex1 subunit of the RNAP, and with the heterolgous gene(s) or xcex1 subunit of the RNAP peferably, two or more vectors may be used. The nucleic acid molecules encoding the xcex1 subunit and the heterologous gene(s) may be in separate vectors for transformation of the host cell. The host cell can optionally be transformed with at least one gene encoding a heterologous transcriptional regulator obtained from the same genus as the source of the heterologous gene. The heterologous gene may comprise multiple genes or operons in the same metabolic pathway.